It is not generally appreciated that diarrheal diseases take an enormous toll in regard to morbidity and mortality in infants and young children in the developing countries of the world. Diarrheal diseases are ranked first among infectious diseases in the number of episodes and deaths in developing countries in Asia, Africa and Latin America. It is estimated that 5–10 million diarrhea-associated deaths occur annually in developing countries, predominantly in infants and young children. Walsh and Warren, N. Engl. J. Med. 301: 967 (1979).
Up until the 1970s, the cause of a major portion of diarrheal illnesses was not known. The rotavirus, discovered in 1973, has since been established as the single most important etiologic agent of severe diarrhea in infants and young children in the developed as well as developing countries, being responsible for approximately 35–50% of such illnesses. In the United States, 90% of infants and young children have experienced a rotavirus infection by the end of their third year of life. Kapikian and Chanock, in “Virology,” 2d ed., Fields et al., eds., Raven Press, New York, pp. 1353–1404 (1990). The disease burden annually in the United States for rotavirus diarrhea in the under-5 year age group is estimated to reach over 1 million cases of severe diarrhea, with approximately 110,00 children hospitalized annually with presumptive rotavirus gastroenteritis, resulting in 583,000 hospital days, and 150 deaths. Matson and Estes, J. Infect. Dis. 162: 598 (1990). In developing countries, rotaviruses are believed to be responsible for the death of over 870,000 infants and young children annually. Institute of Medicine, in “New Vaccine Development. Establishing Priorities. Diseases of Importance in Developing Countries,” Nat'l Academy Press, Washington, D.C. , II: 308–318 (1986).
Rotaviruses are classified as a genus in the family Reoviridae. Rotaviruses are about 70 nm in diameter and have a doubled layered icosahedral protein capsid that consists of an inner and outer layer. Within the inner capsid is the core that contains the 11 segments of the double-strand RNA genome. Rotaviruses are classified serologically into seven distinct serogroups, A–G, and within each group are further divided into serotypes. Only Group A, B and C rotaviruses have been found in humans, and Group A viruses have clearly been established as causing significant disease. Group B viruses have been associated with epidemics predominantly in adults in China, and Group C viruses have been only sporadically isolated from children with diarrhea, and the clinical significance remains unclear. Only one non-A rotavirus, a Group C porcine strain, has been successfully cultivated. Within Group A rotaviruses, ten serotypes associated with human infection have been identified, of which four (numbered 1, 2, 3 or 4) are regarded as epidemiologically important.
Group A rotaviruses possess two outer capsid proteins that function as independent neutralization antigens, namely, VP4 (encoded by genome segment 4) and VP7 (encoded by genome segment 7, 8 or 9 depending on the strain) (Hoshino et al., Proc. Natl. Acad. Sci. USA 82:8701–8704 (1985) and Offit et al., J. Virol. 57:376–378 (1986)). As a consequence a binomial nomenclature is now used to designate rotaviruses. This nomenclature includes designation of VP4 and VP7 rotaviruses. Although initially VP7 was thought to be the dominant neutralization antigen, recent studies have shown that VP4 is as effective as VP7 in inducing neutralizing antibodies following infection of experimental animals (Hoshino et al., J. Virol. 62:744–748 (1988) or susceptible infants or young children (Flores et al., J. Clin. Microbiol. 27:512–518 (1989)). Also, antibodies to VP4 or VP7 are independently associated with resistance of gnotobiotic piglets to experimental challenge with virulent rotavirus (Hoshino et al., J. Virol. 62:744–748 (1988)).
Strategies for the development of rotavirus vaccines have, to date, been based on a “Jennerian” approach, which takes advantage of the antigenic relatedness of human and animal rotaviruses and the diminished virulence of animal rotavirus strains for humans. Kapikian et al., in Vaccines 88, Chanock et al. (eds.), p. 151–159, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1987). Three candidate live oral rotavirus vaccines that have been evaluated in various parts of the world were developed using this approach, where an antigenically-related live virus derived from a nonhuman host is used as a vaccine for immunization against its human virus counterpart. These vaccines contained bovine rotavirus strain NCDV (RIT4237) (VP7 serotype 6) (Vesikari et al., Lancet 2:807–811 (1983)), bovine rotavirus strain WC3 (VP7 serotype 6) (Clark et al., Am. J. Dis. Child. 140:350–356 (1986)), or rhesus monkey rotavirus (RRV) strain MMU18006 (VP7 serotype 3) (Kapikian et al., Vaccines 85, Eds. Lerner et al., Cold Spring Harbor Laboratory, N.Y., pp. 357–367 (1985)). The protective efficacy of the monovalent bovine or simian rotavirus vaccines has proved to be variable. This can be attributed to the fact that the target population of two- to five-month-old infants characteristically developed a homotypic immune response following vaccination (Kapikian et al., Adv. Exp. Med. Biol., 327:59–69 (1992), Bernstein et al., J. Infect. Dis. 162:1055–1062 (1990), Green et al., J. Infect. Dis. 161:667–679 (1990), and Vesikari, Vaccine 11:255–261 (1993)). As a consequence, a modified “Jennerian” approach was developed for the formulation of a tetravalent vaccine. This involved replacing the VP7 gene of the animal (rhesus or bovine) rotavirus by the corresponding gene of each of the human rotaviruses of major clinical importance except in the case of rhesus rotavirus which did not require a serotype 3 VP7 substitution because this simian virus has a serotype 3 VP7. (Kapikian et al., Adv. Exp. Med. Biol., 327:59–69 (1992), Midthun et al., J. Virol. 53:949–954 (1985), Midthun et al., J. Clin. Microbiol. 24:822–826 (1986), and Clark et al., Vaccine 4:25–31 (1986).)
A “non-Jennerian” approach to vaccination has also been considered. Recently, naturally-attenuated strain of human rotavirus, M37 [VP4:2;VP7:1], was evaluated as a vaccine candidate in two- to six-month-old infants. It induced an immune response that was primarily strain-specific (Flores et al., Lancet 2:330–334 (1990) and Vesikari et al., Pediatr. Infect. Dis. 10:912–917 (1991)). However, it failed to induce significant protection against rotavirus diarrhea. Matsuno et al., Virus Res. 7:273–280 (1987)) have reported the selection of a ca human rotavirus strain IGV-80-3 (VP7 serotype 1) which grew efficiently at 25° C., but further characterization of the strain has not been provided.
VP7 is the only relevant rotavirus protective antigen present in candidate vaccines that are currently being evaluated for protective efficacy in humans. This is because these vaccines contain the VP4 of an animal rotavirus or a naturally-attenuated human rotavirus that is not related antigenically to the VP4 of any of the clinically important human rotaviruses.
Although the quadrivalent rhesus rotavirus vaccine provides resistance to serious rotavirus diarrheal disease in 80% of instances (Dennehy et al., Abst. No. 1052, APS-SPR (1994)) there is an urgent need for a more potent vaccine that provides close to complete protection against serious rotavirus diarrhea. In order to achieve this goal it will be necessary to incorporate both protective antigens (i.e., VP4 and VP7) of clinically important rotavirus serotypes into the vaccine. The level of attenuation of the live virus should be sufficiently balanced such that it is capable of propagating to levels adequate for inducing a protective immune response while restricting replication to a level that the virus does not cause clinical disease in the immunized individuals. Quite surprisingly, the present invention fulfills this and other related needs.